U.S. patent application number 15/478924 was filed with the patent office on 2017-10-05 for system, device and method for electroporation of cells.
The applicant listed for this patent is CyteQuest, Inc.. Invention is credited to Thomas N. Corso.
Application Number | 20170283761 15/478924 |
Document ID | / |
Family ID | 59958597 |
Filed Date | 2017-10-05 |
United States Patent
Application |
20170283761 |
Kind Code |
A1 |
Corso; Thomas N. |
October 5, 2017 |
System, Device and Method for Electroporation of Cells
Abstract
A system, device and method for electroporation of living cells
and the introduction of selected molecules into the cells utilizes
a fluidic system where living cells and biologically active
molecules flow through a channel that exposes them to electric
fields, causing the molecules to be transferred across the cell
membrane. The device is structured in a manner that allows precise
control of the cells location, motion, and exposure to electric
fields within the flow channel device. The method is particularly
well suited for the introduction of DNA, RNA, drug compounds, and
other biologically active molecules into living cells.
Inventors: |
Corso; Thomas N.; (Groton,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CyteQuest, Inc. |
Ithaca |
NY |
US |
|
|
Family ID: |
59958597 |
Appl. No.: |
15/478924 |
Filed: |
April 4, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62318022 |
Apr 4, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/46 20130101;
C12M 35/02 20130101; C12N 15/87 20130101; C12M 27/00 20130101; C12N
13/00 20130101 |
International
Class: |
C12M 1/42 20060101
C12M001/42; C12N 13/00 20060101 C12N013/00; C12N 15/87 20060101
C12N015/87; C12M 1/02 20060101 C12M001/02; C12M 1/34 20060101
C12M001/34 |
Claims
1. A device capable of inserting a biologically active molecule
into a living cell comprising: a fluid channel comprising a fluid
input and a fluid output configured to allow a fluid flow
comprising living cells and biologically active molecules through
the channel; and a first electrode and a second electrode on
opposite sides of the fluid channel to which electrical potentials
can be applied to form an electric field directed across the fluid
channel, wherein the distance between the first and second
electrodes enables the cells to pass through the space between the
electrodes in a single layer so a living cell in the fluid flow is
maintained in a similar position as other living cells in the fluid
flow as they pass through the electric field between the first and
second electrodes in a manner that prevents one living cell from
shielding another living cell from the applied electric field,
wherein the strength of the electric field to which the living cell
is exposed is sufficient to form pores within the membrane of the
living cell through which the biologically active molecule can
traverse the cell membrane, but not lyse the living cell.
2. The device of claim 1, wherein the distance between the first
and second electrodes is less than about 100 micrometers.
3. The device of claim 1, wherein the first and second electrodes
are formed by coating the channel on opposite sides with
electrically conducting layers.
4. The device of claim 1, wherein the channel and the electrodes
comprise an optically transparent material to allow observation of
the motion of the living cells in the fluid channel of the
device.
5. The device of claim 1, wherein the electrodes are patterned to
create spatially varying electric fields or the applied electrical
potential is held constant in time or pulsed.
6. The device of claim 1, wherein the dimensions of the channel are
varied in such a way that the velocity of the fluid flow containing
cells varies as a function of the height and width of the
channel.
7. A device capable of inserting a biologically active molecule
into a living cell comprising: a fluid channel comprising at least
two fluid inputs and a fluid output configured to allow a fluid
flow comprising two or more laminar sheath fluid streams of living
cells and biologically active molecules through the channel; and a
first electrode and a second electrode on opposite sides of the
fluid channel to which electrical potentials can be applied to form
an electric field directed across the fluid channel, wherein the
dimensions of the fluid channel and the two or more laminar sheath
fluid streams are sufficient to force the cells to pass through the
space between the electrodes in a single layer so a living cell in
the fluid flow is maintained in a similar position as other living
cells in the fluid flow as they pass through the electric field
between the first and second electrodes in a manner that prevents
one living cell from shielding another living cell from the applied
electric field, wherein the strength of the electric field to which
the living cell is exposed is sufficient to form pores within the
membrane of the living cell through which the biologically active
molecule can traverse the cell membrane, but not lyse the living
cell.
8. The device of claim 7, wherein the dimensions of the laminar
sheath fluid stream containing the living cells between the first
electrode and second electrode is less than about 100
micrometers.
9. The device of claim 7, wherein the first and second electrodes
are formed by coating the channel on opposite sides with
electrically conducting layers.
10. The device of claim 7, wherein the two or more fluid streams
comprise different electrical conductivities.
11. The device of claim 7, wherein the two or more fluid streams
comprise a different chemical composition.
12. The device of claim 7, wherein the electrodes are patterned to
create spatially varying electric fields or the applied electrical
potential is held constant in time or pulsed.
13. The device of claim 7, wherein the channel comprises a
transparent region through which cells can be observed by optical
imaging.
14. The device of claim 7, wherein the dimensions of the channel
are varied in such a way that the velocity of the fluid flow
containing cells varies as a function of the height and width of
the channel.
15. The device of claim 7, wherein a first one of the two or more
fluid streams comprises mammalian living cells and a second one of
the two or more fluid streams comprises biological molecules to be
transported across the cell membranes.
16. A method for inserting a biologically active molecule into a
living cell comprising: flowing fluid comprising living cells and
biologically active molecules through a channel between two
electrodes capable of generating an electric current, each
electrode disposed on opposite sides of the channel; passing the
cells through a space between the two electrodes in a single layer
so a living cell in the fluid flow is maintained in a similar
position as other living cells in the fluid flow as they pass
between the two electrodes; and applying an electric voltage across
the two electrodes while the single layer of living cells is
passing between the two electrodes in a manner that prevents one
living cell from shielding another living cell from the applied
electric field, wherein the strength of the electric field to which
the living cell is exposed is sufficient to form pores within the
membrane of the living cell through which the biologically active
molecule can traverse the cell membrane, but not lyse the living
cell.
17. The method of claim 16, wherein the living cells comprise
mammalian cells.
18. The method of claim 16, wherein the biologically active
molecules are chosen from among the categories of nucleic acids,
drug molecules, and other biologically active molecules.
19. The method of claim 16, further comprising monitoring the
electrical current passing between the two electrodes to provide
information about living cell modifications.
20. The method of claim 24, wherein the two electrodes are
separated by a distance of approximately 1% to 50% greater than the
size of the living cells.
Description
CROSS REFERENCE
[0001] This application claims the benefit of the filing date of
U.S. Provisional Patent Application Ser. No. 62/318,022 filed Apr.
4, 2016, which is hereby incorporated by reference in its
entirety.
FIELD
[0002] The present disclosure relates to methods of molecular
biology, and more specifically a system, device, and method for the
electroporation of cells.
BACKGROUND OF THE INVENTION
[0003] In medicine and biomedical research, there is motivation to
insert biologically active molecules into selected living cells.
These molecules could be drugs to treat specific diseases, but an
important application is the insertion of nucleic acid molecules
such as DNA and RNA, which is typically called transfection or
transformation. The inserted nucleic acid molecules can serve as a
vaccine, can enable the cellular production of specific proteins,
or can be used to reprogram the human immune system cells to attack
tumors or disease causing cells. In such applications it is
critical to insert sufficient DNA or RNA into a cell without doing
damage that could kill the cell. Control of the process is
important and process parameters generally differ for different
types of cells.
[0004] A method known as electroporation, or
electropermeabilization, has been used for decades as an approach
to electrically open pores in cell membranes to allow the passage
of molecules into the cells. In this method, electric fields are
created by applying high voltage electrical pulses to electrodes
inserted in a liquid container containing cells suspended in a
liquid solution that contains the molecules to be inserted into the
cells. The applied high-voltage pulses create transient pores in
the cell membrane that allow molecules to pass into the cells.
However, the open pores also allow the cell contents to escape with
negative consequences for the health of the cell. The pulse
voltage, number of pulses and pulse duration are among the
parameters empirically varied to optimize the efficiency of
molecular insertion and cell survival. However, current devices are
limited in that molecules are exposed to a large range of electric
fields, often causing biologically active molecules to not transfer
efficiently and/or many cells to be damaged or killed during
electroporation. Current devices also lack process variability and
cannot be optimized for various cell types and biologically active
molecules. Furthermore, current devices have limited throughput.
These drawbacks have limited the widespread application of this
method.
[0005] Some improvement in throughput has been made by flowing a
solution with living cells and biologically active molecules
through the container with electrodes. For example, a publication
by Choi et al. (2010) proposed a high-throughput
microelectroporation device for introducing chimeric antigen
receptor to human T cells to redirect their specificity. In
addition, U.S. Pat. Nos. 4,752,586; 5,612,207; 6,074,605 and
6,090,617 refer to electroporation with flow for processing large
number of cells. These devices introduce flow to fill and empty the
electroporation chamber, but the living cells are still subjected
to various electric fields depending on their distance from the
electrodes during flow through the chamber. Thus, the efficiency of
molecular transformation as well as the potential lysing of the
cells remains a problem.
[0006] U. S. Patent Application Publication 2014/0066836, discloses
an electroporation device that includes movable electrodes in order
to achieve a more specific spatial configuration between the
electrodes and the cells. However, the cells reside in a bulk
solution in the device or in vivo. Thus, the quantity of cells that
are exposed to the precise field strength is limited.
[0007] There are also additional practical limitations of the
current electroporation methods. For example, high voltages are
required and it is often necessary to pulse the voltage in order to
allow the cells to recover in between voltage exposure. Also,
current devices permit cells to be porated only in a single,
homogenous fluid environment. In addition, current devices preclude
the ability to optically or electrically monitor the cell motion
and electroporation process while it is occurring.
[0008] Currently, the art lacks a system, method and device for the
introduction of biologically active molecules into flowing living
cells by electroporation in a manner that allows control of the
living cells' location, motion, local chemical environment and
exposure to electric fields.
SUMMARY
[0009] In accordance with one aspect of the present disclosure
there is provided a device capable of inserting a biologically
active molecule into a living cell including a fluid channel
including at least one fluid input and a fluid output configured to
allow a fluid flow including living cells and biologically active
molecules through the channel; and a first electrode and a second
electrode on opposite sides of the fluid channel to which
electrical potentials can be applied to form an electric field
directed across the fluid channel, wherein the distance between the
first and second electrodes enables the cells to pass through the
space between the electrodes in a single layer so a living cell in
the fluid flow is maintained in a similar position as other living
cells in the fluid flow as they pass through the electric field
between the first and second electrodes in a manner that prevents
one living cell from shielding another living cell from the applied
electric field, wherein the strength of the electric field to which
the living cell is exposed is sufficient to form pores within the
membrane of the living cell through which the biologically active
molecule can traverse the cell membrane, but not lyse the living
cell.
[0010] In accordance with another aspect of the present disclosure
there is provided a device capable of inserting a biologically
active molecule into a living cell including a fluid channel
including a fluid channel including at least two fluid inputs and a
fluid output configured to allow a fluid flow including two or more
laminar sheath fluid streams of living cells and biologically
active molecules through the channel; and a first electrode and a
second electrode on opposite sides of the fluid channel to which
electrical potentials can be applied to form an electric field
directed across the fluid channel, wherein the dimensions of the
fluid channel and the two or more laminar sheath fluid streams are
sufficient to force the cells to pass through the space between the
electrodes in a single layer so a living cell in the fluid flow is
maintained in a similar position as other living cells in the fluid
flow as they pass through the electric field between the first and
second electrodes in a manner that prevents one living cell from
shielding another living cell from the applied electric field,
wherein the strength of the electric field to which the living cell
is exposed is sufficient to form pores within the membrane of the
living cell through which the biologically active molecule can
traverse the cell membrane, but not lyse the living cell.
[0011] In accordance with another aspect of the present disclosure
there is provided a method for inserting a biologically active
molecule into a living cell including flowing fluid including
living cells and biologically active molecules through a channel
between two electrodes capable of generating an electric current,
each electrode disposed on opposite sides of the channel; passing
the cells through a space between the two electrodes in a single
layer so a living cell in the fluid flow is maintained in a similar
position as other living cells in the fluid flow as they pass
between the two electrodes; and applying an electric voltage across
the two electrodes while the living cell is passing between the two
electrodes in a manner that prevents one living cell from shielding
another living cell from the applied electric field, wherein the
strength of the electric field to which the living cell is exposed
is sufficient to form pores within the membrane of the living cell
through which the biologically active molecule can traverse the
cell membrane, but not lyse the living cell.
[0012] These and other aspects of the present disclosure will
become apparent upon a review of the following detailed description
and the claims appended thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a cross sectional schematic view of a portion of a
fluid channel device in accordance with an embodiment of the
present disclosure;
[0014] FIG. 2 is a cross sectional schematic view of a fluid
channel device including a fluid channel system, multiplicity of
fluid inputs, output, and a pair of electrodes;
[0015] FIG. 3 shows an embodiment of a device constructed with
three layers;
[0016] FIG. 4 shows a plan view schematic of a flow channel device
with variable width in a direction orthogonal to the direction of
flow;
[0017] FIG. 5 shows a schematic of a system for control of fluid
flow and electrical voltage and optically and electrically
monitoring controlled electroporation;
[0018] FIG. 6 shows a cross-sectional schematic view of an
embodiment of the device; and
[0019] FIG. 7 shows a side view of a device having a variable
height channel in chip.
DETAILED DESCRIPTION
[0020] The present disclosure relates to a system, method and
device for the introduction of a biologically active molecule into
a living cell by electroporation. The disclosure allows for
monitoring and controlling cell location, motion, and exposure to
electric fields between electrode pairs within the device such that
each cell is exposed to similar electrical and chemical conditions
during electroporation. In an embodiment, an electroporation device
contains a fluid channel flanked by two electrodes on opposite
sides of the channel to which electrical potentials can be applied
to create an electric field across the channel between the
electrode pair. The dimensions of the fluid channel combined with
the characteristics of the fluid flow provide sufficient control to
maintain the individual living cells within the fluid flow at
similar positions with respect to proximity to the passing
electrode pair. As the living cells flow through the channel
between the electrodes the distance from the cell to each electrode
is held to be nearly constant and in a manner that prevents one
living cell from shielding another living cell from the applied
electric field. Typically, the cell flow is one layer thick in the
channel dimension between the opposing electrode pairs so that the
cells are independently exposed to the same electrical current
formed in the channel when passing between the electrode pairs. The
channel has no restriction on distance in the other two dimensions
of channel length and opposing channel walls not flanked by the
electrodes. The cells flow through the channel at a set flux, and
these features enable the user to apply precise electric fields to
the cell. The strength of the electric field is strong enough to
form pores within the membrane of the living cell through which
biologically molecules can traverse the cell membrane, but weak
enough to not lyse the cell.
[0021] The device includes one or more fluid inputs and at least
one fluid output. When the device includes a single fluid input a
single laminar fluid stream is created. The single fluid stream
contains living cells in combination with biologically active
molecules for introduction of the biologically active molecule into
the living cell by electroporation. Suitable spacing between the
electrodes includes about 2 to 5 times larger than the diameter of
the cell, or smaller than approximately two times the typical cell
diameter, forcing the cells to pass through the space between the
electrodes in a single layer. The living cells in the single fluid
flow are maintained in a similar position as other living cells as
they pass through the electric field between the electrode pairs so
each cell is exposed to similar electrical and chemical conditions
during electroporation. Suitable distance between the electrodes of
an electrode pair includes a range of from about 50 micrometers to
about 100 micrometers, or less than about 100 micrometers.
[0022] When the device includes at least two fluid inputs multiple
laminar sheath fluid streams are created. Each fluid input can
accept a separate fluid stream. For example, one stream contains
living cells and another contains the biologically active
molecules. Thus, the living cells and the biologically active
molecules flow through separate fluid inputs into the channel. The
streams are separated by laminar sheath flow. The dimensions of the
fluid channel are constructed to accommodate the laminar flow
separated streams so that living cells contained in the fluid flow
are maintained in a similar position as other living cells as they
pass through the electric field between the electrode pairs. In a
system with multiple sheath flows, the sheath flows separate the
cells from the electrode and channel walls with a constant spacing
controlled by the flow rates. The multiple sheath flow devices
allow the chemical composition of the fluid on opposite sides of
the cell to differ permitting an efficient electrical drive of
charged molecules such as DNA and RNA into the cells. The flow of
liquid through the channels can be unvarying in time, which
simplifies the process and assures that all cells experience the
same combination of conditions during electroporation as they pass
through the flow channel. Suitable distance between the boundaries
of the sheath flow containing the living cells between paired
electrodes includes from about 50 micrometers to about 100
micrometers; less than about 100 micrometers; about 2 to 5 times
larger than the diameter of the cell; or smaller than approximately
two times the typical cell diameter, forcing the cells to pass
through the space between the electrodes in a single layer. The
device can contain one or more fluid outputs. In this embodiment,
the distance between the electrode pairs can be greater than that
noted above since the living cells in the sheath stream are
maintained in a similar position as other living cells as they pass
through the electric field between the electrode pairs by the
adjacent sheath flows. A suitable distance between the electrodes
of an electrode pair includes from about 50 micrometers to about
500 micrometers. Another advantage of an embodiment of the
disclosure is that the user can manipulate the chemical and
electrical properties of the environment at different positions
along the length of the channel. Furthermore, an embodiment of the
disclosure allows the user to monitor various properties of the
cells and/or the solution in order to modify and optimize the flow
and voltage parameters in real time.
Channel
[0023] FIG. 1 shows a cross-sectional schematic view of an
embodiment of the device. A flow channel 106 lies between two chips
100. A positive electrode 101 lies on an inner flow channel surface
of upper chip 100 opposite a negative electrode 102 which lies on
an inner flow channel surface of lower chip 100. A liquid stream
106 of buffer containing cells 108 and nucleic acids or other
biological molecules 110 to be electroporated flows between lower
chip 100 and upper chip 100.
[0024] By limiting the gap dimension between electrodes to be about
2 to 5 times larger than the diameter of the cell or less than
approximately two times the cell diameter, there is not enough
physical space for more than one cell in the flowing stream to be
located in the channel gap between the electrodes. This controlled
gap spacing as well as operating in the laminar flow regime (no
turbulent flow) allows for controlled positioning of a single given
cell between the electrodes in this one plane. While the flow
channel is narrow in proximity to the electrodes, the channel can
be made as wide as necessary in the orthogonal dimension to achieve
the desired flow rate of the cells through the channel. Similarly,
the length of the channel has no restrictions. Control of the
distance between the electrodes allows each cell to be isolated or
held in a similar position relative to the electrodes. Thus, each
cell is essentially subjected to a similar electrical and chemical
environment while, at the same time, high total cell throughput is
possible. In one embodiment of the device, the channels can be
manufactured so that distance between the chips 100, and thus the
electrodes 101 and 102, can be adjusted to accommodate different
types and sizes of living cells. The chips 100 on which the
electrodes 101 and 102 are mounted can be made of any nonconductive
or electrically insulating material, such as glass, plastic or
optically transparent material.
[0025] FIG. 2 shows a cross-sectional schematic view of an
embodiment of the device. A flow channel 107 lies between chips 100
with patterned electrodes; a positive electrode 101 lies opposite a
negative electrode 102. Fig. A lower liquid stream 103 of a high
conductivity buffer containing nucleic acids or other biological
molecules to be electroporated flows adjacent the negative
electrode 102. An upper liquid stream 104 of a high conductivity
buffer flows adjacent the positive electrode 101. A middle liquid
stream 105 of a low conductivity buffer containing cells 108 to be
electroporated flows between lower liquid stream 103 and upper
liquid stream 104. The upper liquid stream 104, middle liquid
stream 105, and lower liquid stream 102 are separated by laminar
flow.
[0026] In FIG. 2, fluid from the inputs 103, 104, and 105 flow
through the channel 107 and exit the device via an output 108 flow.
Spacers 106 are used to change direction of the flow. The flow
channel can be made in various geometries and have either a
constant or variable width. For example, FIG. 4 illustrates a top
view of a device having a variable width channel, wherein the
channel is narrower as it passes by the electrodes 101, and wider
in between electrodes. Each upper electrode 101 is paired with a
lower electrode 102 (not shown). Thus, spacer outline 106
illustrates an area of fast flow across the electrodes 101 and an
area of slow flow in between the electrodes 101. The stream from an
upper liquid outlet 104 and the stream from a middle liquid outlet
105 are separated by laminar flow and exit via outlet 108.
[0027] FIG. 6 shows a cross-sectional schematic view of an
embodiment of the device. The flow channel 207 lies between chips
200 with patterned paired electrodes; two positive electrodes 101
lie opposite two negative electrodes 102. The distance between the
two electrodes is selected to be slightly larger than the diameter
of the living cells flowing in fluid input 205. While the flow
channel is narrow in proximity to the electrodes, the channel can
be made as wide as necessary in the orthogonal dimension to achieve
the desired flow rate of the cells through the channel. Similarly,
the length of the channel has no restrictions. Control of the
distance between the paired electrodes allows each cell to be
isolated or held in a similar position relative to the paired
electrodes, taking into account the laminar flow established by
fluid inputs 203, 204, and 205. Thus, each cell is essentially
subjected to a similar electrical and chemical environment while,
at the same time, high total cell throughput is possible. Fluid
from the inputs 203, 204, and 205 flow through the channel 207 and
exits the device via an outlet flow 208. Spacers 206 are used to
change direction of the flow.
[0028] The flow channel can be made in various geometries and have
either a constant or variable height. For example, FIG. 7
illustrates a side view of a device having a variable height
channel between chips 200, wherein the channel is lower as it
passes by the paired electrodes 501, 502, and taller in between the
paired electrodes 501, 502. Thus, the channel illustrates an area
of fast flow across the electrode pairs and an area of slow flow in
between the electrode pairs. The stream from an upper liquid inlet
205 and lower liquid outlet 206 changes flow direction by spacers
504.
Fluid Inputs and Streams
[0029] The fluid can flow through the channel at a rate of 0.1
cm/s, with a relevant range of flow rate between 0.001 cm/s and 10
cm/s. The volume of fluid flowing through the channel relates to
the cross sectional area of the flow channel. For example, for a
channel 2 cm wide and 100 micrometer high the volumetric flow rates
would be in the range of from about 0.2 microliters/s to 2
milliliters/s.
[0030] The device permits the use of multiple inputs of fluid
through slits in the channel device to provide flow with different
layers of solution composition. The flow rate of two or more fluid
streams into the channel can be controlled to create a sheath flow.
In one embodiment of the device, the channel 107 (FIG. 2) delivers
a low conductivity buffer containing living cells to be
electroporated through a liquid sheath. An optional channel 104, on
the same side of the device as channel 105, delivers a high
conductivity buffer. Another channel 103, located on the opposite
side of the device, also delivers high conductivity buffer; in FIG.
2 this buffer contains the biologically active molecules that are
to be inserted into the living cells. Various streams of cells or
molecules enter the channel via these inputs, and these streams can
have the same or different flow rates. If desired, streams with
different flow rates adopt laminar flow through the channel. Thus,
the streams flow in parallel through the channel and remain largely
separated, mixing slowly only through diffusion. In this manner,
individual cells in the stream of living cells can be isolated
between the electrode pair by the laminar flow of the adjacent
fluid streams.
[0031] The use of multiple inputs of fluid can prevent various
types of fouling or contamination. For example, the molecules or
nucleic acids to be inserted into the cells can exist in a separate
solution from the cells. This can be useful because certain
molecules, like RNA, may not be stable in the vicinity of living
cells due to enzymes on the cell surface or cell culture media.
Also, it is known that degradation of the electrodes can result in
the release of contaminants that are toxic to cells. The separate
fluid layers ensure that the cells remain free from contaminants
from the electrodes. Further, the cells themselves are kept out of
contact with both the surface of the chip and the electrodes
preventing possible contamination.
[0032] Alternatively, an embodiment of the device can contain a
single fluid input through which a homogenous solution of cells and
biologically active molecules enters the channel. The stream
consists of a conductive buffer solution containing the
biologically active molecules that are to be inserted into the
living cells. The biologically active molecules are chosen from
among the categories of nucleic acids, drug molecules, and other
biologically active molecules. Compared to the device having
multiple inputs, this might be advantageous in that there is a
greater opportunity for the cells and the biologically active
molecules to come into contact with one another and could increase
the efficiency of transformation.
[0033] In one embodiment, the inputs 104, 105 introduce fluid
streams to the channel so that the streams turn at an angle before
flowing in between the electrodes. In FIG. 2, this angle is shown
as 90.degree., but the angle can be any angle including 0.degree..
In this case, spacer 106 helps to direct the flow from the inlets
104, 105.
[0034] Similarly, in one embodiment of the disclosure, the flow
turns a corner before exiting the device through the output 108. In
FIG. 2, this angle is 90.degree., but this angle can be any angle,
including 0.degree.. In this case, spacer 106 helps to direct the
flow to the outlet.
[0035] Fluid streams interface to the device via interconnects, a
manifold, or discreet fluid path connections. The manifold serves
to reformat the tubing or conduits into the receiving slit-port of
the device (103, 104, or 105 in FIG. 2). The manifold may have
surface area changes for this purpose. The manifold may have
features to enhance mixing or maintain laminar flow
characteristics.
[0036] The manifold may be machined, molded, casted, or the like.
The manifold may also be fabricated as part of the device or bonded
to the device via a permanent or non-permanent bond. Sealing
between the manifold and the device may be hermetic,
compression-based, O-ring-based, gasket-based, adhesion-based,
fused, luer locked, flat bottom compression-based, tapered
ferrule-based, frusto-conical compression-based, or the like.
[0037] Cells and the bioactive materials may be presented to the
device via several approaches. They may be injected via a robotic
fluid handling platform or injection system or connected via
biocompatible containers. Bioprocess containers include polymer
bags, T-flasks, conical tubes, media bottles, well plates, or the
like. These vessels may be one time use or reusable when proper
sterilization is performed. Connections to the fluid delivery path
may be achieved by compression seals, threaded connections,
clamping compression, luer lock mechanisms, O-ring seals, friction
seals, gaskets seals, clamping, or similar connections. In the case
of pneumatic displacement, the container itself may be pressurized
or be contained inside a pressurized vessel.
[0038] In another embodiment, the cells may be presented to the
device by custom cartridges that interface to the pumping or fluid
manipulation system.
Fluid Output
[0039] The fluid outlet 108 is shown in FIG. 2. After
electroporation, a mixture of all of the fluid streams can leave
the device via this outlet. The solution may be transferred to
sterile polymer bags, T-flasks, conical tubes, media bottles, well
plates, or the like and allowed to recover at 37.degree. C. The
cells may then be re-suspended in standard tissue culture medium
and plated for immediate use in cellular assays, cryopreserved for
future use, or used as desired.
Electrodes
[0040] The separation between electrodes in accordance with the
present device is small therefore requiring an applied voltage of
only a few volts to perform the electroporation. This is in
contrast to the need for voltages up to several thousand volts that
are normally required for prior art electroporation. For example,
it is known in the literature that a transmembrane electric field
of less than 1 kV/cm is required to porate the cell membrane
(Weaver and Chizmadzhev, 1996). However, for a distance between the
electrode pairs of 100 micrometers, this requires approximately a 5
V potential difference to porate an average mammalian cell in
accordance with the present device. Suitable voltage differences
across a living mammalian cell include the following range: 0.1 V
to 10 V. For example, for a distance between the electrodes of 100
micrometers this range corresponds to an electric field of 10 V/cm
to 1000 V/cm.
[0041] The flow channel can have one or several electrically
independent electrode pairs. For example, in FIG. 4, four sets of
electrode pairs 101 are shown. Connections to the electrodes are
made by using clips or conduction adhesive to connect these to a
variable-voltage power supply or batteries with a voltage divider.
An ammeter can be used to monitor the current flowing between
electrodes for monitoring and controlling the process.
[0042] The electrodes can apply either a constant or pulsating
voltage through the channel. If a pulsating voltage is desired,
pulse duration of from about 0.01 millisec to about 100 millisec is
suitable. The plurality of electrode pairs can be patterned to
create spatially varying electric fields. The electrodes may be
patterned by the use of a photomask in the photolithographic
process or by a shadow mask in the sputtering or deposition
process. Patterning allows for the fabrication of electrodes with
varying geometric shape.
Manufacturing the Device
[0043] One embodiment of the device is constructed from a
three-layer stack of polymer substrates or plastics as shown in
FIG. 3. All three layers are laser cut with a small beam spot, high
resolution CO.sub.2 laser. The layers on which the electrodes are
fixed are cut from 1 mm thick acrylic slabs, creating opposite
surfaces of the channel. A middle layer 106 defines the distance
between the electrode pairs 101, 102. In the embodiment shown in
FIG. 3, the three dimensions of the layers are the same. Although
it is most practical for the layers to be the same in dimensions in
the plane that the stream flows, these dimensions can be different
from one another. One way to manufacture these layers is to use a
laser to cut acrylic pieces to microscope format 25.times.75 mm,
add fluid inlet slits or ports 103, 104, 105 to chips 100,
respectively and add alignment holes 109 to facilitate assembly. A
thin film electrode (50 nm) of a gold-palladium (Au/Pd) mixture is
deposited by physical vapor deposition on the inside surface of
each acrylic piece. The 100-micron thick middle layer 106 polymer
film with medical adhesive on each side is cut to shape and also
receives the corresponding alignment holes via the laser cutting
process. After laser cutting, the three pieces are placed on a jig
containing alignment pins corresponding to the alignment holes in
each layer. The sandwich assembly is then compression-bonded in a
press. This two-step process of laser cutting and compression
assembly is amenable to mass production and allows for a
cost-effective consumable to be created. The process can be used to
manufacture hundreds of thousands of devices per year. This is in
contrast to many other types of prior art non-electroporation
microfluidic devices that typically require expensive capital
equipment and a large number of chemical processing steps.
[0044] Alignment of the device layers may be conducted by optical
positioning or a physical means such as datum pads, alignment pins,
or structures. The device layers may have receiving features for
use with a jig alignment piece or system. Alternatively, the
alignment features may reside in the device layers as so no jig or
peripheral alignment system is necessary. These may include
pin-like structures or features that snap together.
[0045] The flow cell could also be produced by an injection molding
process, where the volume can scale to millions of single-use
devices per year, using one injection molding press with a
multi-cavity mold.
[0046] This disclosure allows for architectures for manufacturing
the device that are readily amenable to injection molding. In this
device, all the layers may be formed via injection molding. The
fluidic channel may be formed in one layer at full depth or,
alternatively, the channel may span two or more layers, where the
full depth is achieved upon assembly. Injection ports may be
created via core pins. Alternatively, the fluid inlets may be added
post molding as a secondary operation or structure. The layers may
be molded from the planar surface or from the edges. Appropriate
and efficient part release from the mold cavity is known in the
art.
[0047] The molded layers may be assembled together through
mechanical connection, adhesion, bonding, welding (including
ultrasonic and laser), fusing, melting, or the like. Additionally,
there may be another material between the layers for connection and
sealing such as, but not limited to, a gasket, O-ring, washer, or
the like. Alternatively, sealing can be achieved through press
tight or bonding features.
[0048] In another embodiment, the fluid channel may reside in one
layer and the opposing sealing structure is a non-injected molded
part such as a film, tape, or planar material containing necessary
fluid inlets.
[0049] In another embodiment, the device may be created by
three-dimensional printing or additive manufacturing processes.
Other fabrication techniques include compression molding, casting,
and embossing.
[0050] In another embodiment, devices are made from glass via
lithography and wet or dry etching. Alternatively, the devices may
be physically machine via computer numeric control (CNC) or
ultrasonic machining.
[0051] In other embodiments, the devices can be made from various
materials, such as, for example, where at least one layer is glass,
where at least one layer is plastic, where one of the layers is
optically transparent, or where the channel material is
electrically insulating.
Manufacturing the Electrodes
[0052] The formation of patterned electrodes on the flow channel
surface can be accomplished with a variety of readily available
techniques. One method is to use the process of sputtering for
deposition of a conducting metallic conducting layer such as gold,
platinum, aluminum, palladium, other metals, or alloys of multiple
metals. Gold-palladium is an example of a metallic alloy that can
be used to compose the electrodes. The electrodes can be made of an
optically transparent material to allow observation of the motion
of the living cells in the fluid channel of the device. To generate
transparent conducting layers, films of indium-tin oxide (ITO) are
frequently used. After metal deposition, these conducting layers
can be patterned by masking and etching to remove material where it
is not wanted in order to form the desired patterned electrode
shapes. Appropriate masks may be formed from photoresist using
common photolithographic exposure processes.
[0053] Another method for forming electrodes is to deposit
electrically conducing films made of metals or other conducting
layers such as ITO. By depositing them through a prepositioned
mask, sometimes called a shadow mask, the films are positioned in
proximity to the surface to be coated so that the conducting layer
reaches the surface only where previously opened regions have been
formed in the mask. In addition, a related technique called
"lift-off" can be used, in which a patterned photoresist layer can
be used to shape the pattern of deposited conducing material.
[0054] The deposition of layers of conducing ink can be performed
by brushing or spraying, followed by heating to form patterned
conducting films.
[0055] These thin film patterning processes are well known to those
skilled in the art. In this case, the thickness of the films is
desired to be in the range of from 5 nm to 5 micrometers, with a
preferred range of from 10 nm to 100 nm.
[0056] In one embodiment of the device, electrodes can be formed by
inlaying wires in grooves formed in the chip (100 in FIG. 1)
instead of affixing the electrodes to the chips. In this
embodiment, grooves are machined into the chip, for example a
plastic chip, and the electrodes are metal. Preferably, the
electrodes are gold or a gold-plated metal. The wire is then glued
into the groove.
[0057] An embodiment of a system includes an electroporation
device, fluid delivery system including a pump, temperature control
and optical and electrical monitor of the cells to obtain real-time
feedback on the cell modification process. Feedback can be obtained
by monitoring the electrical current passing between the two
electrodes to provide information about living cell modifications,
imaging of the living cells to provide information about living
cell modifications or monitoring fluorescence of the living cells
to provide information about living cell modifications.
[0058] An embodiment includes a system for inserting a biologically
active molecule into a living cell includes an electroporation
device capable of performing a cell modification process including
inserting a biologically active molecule into a living cell
contained in a fluid flow by flowing fluid including living cells
and biologically active molecules through a channel between two
electrodes, each electrode disposed on opposite sides of the
channel; passing the cells through a space between the two
electrodes in a single layer so a living cell in the fluid flow is
maintained in a similar position as other living cells in the fluid
flow as they pass between the two electrodes; and applying an
electric voltage across the two electrodes while the living cell is
passing between the two electrodes in a manner that prevents one
living cell from shielding another living cell from the applied
electric field, wherein the strength of the electric field to which
the living cell is exposed is sufficient to form pores within the
membrane of the living cell through which the biologically active
molecule can traverse the cell membrane, but not lyse the living
cell; a fluid delivery system including a fluid source and a fluid
pump in fluid communication with the electroporation device; an
electrical current source in electrical communication with the pair
of electrodes; a temperature control in thermal communication with
in the fluid flow; and an optical and electrical monitor of the
living cell capable of obtaining real-time feedback on the cell
modification process.
[0059] One advantage to the electroporation device over the prior
art is the ability to optically and electrically monitor the cells
to obtain real-time feedback on the cell modification process. FIG.
5 illustrates one embodiment of the device; this is a microfluidic
electroporation system with an observation microscope 605.
Accordingly, the fluid flow controller 601 or voltage controller
606 can be adjusted as required to optimize the process efficiency
and cell viability. In this embodiment, the microscope is
positioned so that it views a reservoir 602 that contains
biologically active material. For example, this could be nucleic
acids. The fluid from input cell reservoir 600 flows through the
channel of the microfluidic electroporation chip 604 and across the
field of view of the microscope 605, and into a cell collection
reservoir 603, thus enabling the user make adjustments as necessary
to improve the efficiency of transformation.
[0060] Temperature control of the solutions or materials in contact
with the fluids may be implemented at any instance(s) in the
system, including heating and cooling. This may include static
control or temperature cycling.
[0061] The device can be interfaced to a fluid delivery system. A
fluid delivery apparatus or pump operating with flow controller 601
is configured to displace, preferably, indirectly displace, the
fluid from the input cell reservoir 600 to establish a fluid flow
within the fluid path. The fluid displacement apparatus is capable
of providing positive and/or negative displacement of the fluid.
The delivery pump includes mechanisms based on peristalsis,
pneumatics (pressure displacement), hydraulics, pistons, vacuum,
centrifugal force, manual or mechanic pressure from a syringe, and
the like. Preferably, the fluid is indirectly displaced by the pump
without the fluid directly contacting any of the moving parts of
the apparatus, such as, for example, a peristaltic pump acting upon
a fluid filled tube. Alternatively, a pneumatic displacement
mechanism may be used where a head pressure displaces liquid from a
pressurized vessel. Conversely, fluid may be directly displaced by
an apparatus, when the fluid is displaced by directly contacting
any of the moving parts of the apparatus, such as, for example, the
plunger of a syringe pump.
[0062] The pump may include a flow sensor for monitoring the flow
rate or the flow sensor may provide closed loop feedback to the
pump control system. The closed loop feedback can ensure accuracy
and reduce pulsing. The pump displaces fluid contained in flexible
tubing to create a fluid stream. The system may operate with an
inline flow sensor configured to directly measure the fluid flow
rate as the fluid passes the sensor. The system includes a feedback
control in communication with the fluid displacement apparatus and
the inline flow sensor. The inline flow sensor measures the flow
and communicates with a feedback control mechanism. Suitable types
of flow sensor mechanisms include thermal pulse, ultrasonic wave,
acoustic wave, mechanical, and the like. The inline sensor may be
mechanical-based, electrically-based, motion-based, or
microelectromechanical systems (MEMS)-based. The sensor mechanism
may be thermal, ultrasonic or acoustic, electromagnetic, or
differential pressure. One example of a sensor suitable for use in
accordance with the present disclosure is a thermal-type flow
sensor where the sensor typically has a substrate that includes a
heating element and a proximate heat-receiving element or two. When
two sensing elements are used, they are preferably positioned at
upstream and downstream sides of the heating element relative to
the direction of the fluid (liquid or gas) flow to be measured.
When fluid flows along the substrate, it is heated by the heating
element at the upstream side and the heat is then transferred
non-symmetrically to the heat-receiving elements on either side of
the heating element. Because the level of non-symmetry depends on
the rate of fluid flow and that non-symmetry can be sensed
electronically, such a flow sensor can be used to determine the
rate and the cumulative amount of the fluid flow. This mechanism
allows the flow to be measured in either direction. In one
preferred embodiment, the temperature sensors and the heating
element are in thermal contact with the exterior of the fluid
transporting tube and as the fluid stream only contacts the
internal surfaces of the tube, the fluid media avoids direct
contact with the sensor and heating elements. This format type
allows highly accurate and highly sensitive flow measurements to be
performed.
Examples
[0063] The disclosure will be further illustrated with reference to
the following specific examples. These examples are given by way of
illustration and are not meant to limit the disclosure or the
claims to follow.
Example 1--Electroporation of Mammalian Cells with a DNA
Plasmid
[0064] This example describes an embodiment where the flow
electroporation device is used to electroporate mammalian cells
with a DNA plasmid. Chinese hamster ovary (CHO-K1) cells (ATCC) are
electroporated with a plasmid that expresses green fluorescent
protein using the flow-through electroporation device described.
Cell viability can be determined based on the uptake of propidium
iodide. The electroporation efficiency can be determined using
fluorescent observation of the number of cells that express the
green fluorescent protein relative to the total number of
cells.
[0065] The cells are cultured in an incubator at 37.degree. C. and
5% CO.sub.2. The cells can be cultured in a synthetic medium, such
as Dulbecco's modified Eagle's Minimum Essential Medium (DMEM,
Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum
(Sigma) and 100 mg/mL streptomycin (Sigma). When the cell
suspension density reaches a certain value, for example,
2.times.10.sup.6 cells/mL, the cell suspension is diluted with
additional culture medium. Prior to introduction into the device, a
10 mL sample of the suspension is centrifuged at 300 g for 5 min.
The supernatant is discarded and the cells are re-suspended in a
low conductivity buffer (described below). The cell suspension
density for electroporation is preferably 1.times.10.sup.8 cells/mL
with a range between 1.times.10.sup.7 and 1.times.10.sup.9
cells/mL.
[0066] The low conductivity buffer is composed of 0.8 mM
Na.sub.2HPO.sub.4, 0.2 mM KH.sub.2PO.sub.4, 0.1 mM
MgSO.sub.4.7H.sub.2O, and 250 mM sucrose, at a pH of 7.4. This
buffer is made by dissolving 0.1136 g of Na.sub.2HPO4, 0.0272 g of
KH.sub.2PO.sub.4, 0.02465 g of MgSO.sub.4.7H.sub.2O, and 85.575 g
of sucrose in 1 liter of water, and subsequent adjustment of the
pH. The sucrose is used to equalize the osmotic pressure of the
buffer with that of the cells. The buffer is filtered with a
0.2-micron membrane and stored at 4.degree. C. The concentrations
of salts in the buffer as described result in a solution with
electrical conductivity of approximately 0.014 S/m. The preferable
range of the electrical conductivity of this buffer is between
1.times.10.sup.-3 and 0.5 S/m.
[0067] The pAcGFP-C1 plasmid (4.7 Kb, Clontech, Mountain View,
Calif.) encodes a green fluorescent protein (GFP) from Aequorea
coerulescens and contains an SV40 origin for replication in
mammalian cells. The GFP protein is excited at 475 nm and emits at
505 nm. The plasmid is amplified in E. coli and purified using the
QiAfilter Plasmid Mega Kit (Qiagen, Valencia, Calif.) according to
the manufacturer's instructions. The plasmid DNA is dissolved in
Tris-EDTA buffer and stored at -20.degree. C. until use. The
plasmid DNA concentration is determined by ultraviolet (UV)
absorbance at 260 nm. Prior to an electroporation experiment, the
plasmid is precipitated with ethanol and resuspended in phosphate
buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM
Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4) buffer with an
electrical conductivity of approximately 1.5 S/m at a concentration
of approximately 40 ug/mL. The range of the electrical conductivity
of this buffer is between 1.times.10.sup.-2 and 10 S/m. The range
of the plasmid concentration is between 0.01 and 100 ug/mL.
[0068] The low electrical conductivity buffer used for the cell
flow inlet 105 (FIG. 2) used in combination with a higher
electrical conductivity buffer (PBS) for the upper and lower sheath
inlets 103 and 104 flow layers (FIG. 2) results in an electric
field that is substantially larger across the cell flow layer for a
given applied voltage. For a typical experiment, the pressure of
each flow is adjusted so that the cell flow layer is approximately
50 microns deep and the upper and lower sheath flow layers are
approximately 25 microns each in depth. The electrical
conductivities of the low and high conductivity buffer are 0.014
S/m and 1.5 S/m, respectively. The electrical resistance of the
sheath layer (for a voltage applied between the two chip surfaces
100 as shown in FIG. 2) is approximately 99% of the total
resistance. This means that if 5 V is applied between the
electrodes on the two chip plates that the electric fields in the
streams adjacent to the electrodes is approximately 9 V/cm while
the stream sandwiched between those two streams is 991 V/cm.
[0069] It is known that a difference of approximately 1 V between
the interior and exterior of a cell will result in the formation of
pores that can allow for the passage of nucleic acid molecules. The
potential difference U across a cell membrane at a point on the
surface of a cell in an external electric field of strength E is
given by U=fER cos .theta., where R is the cell radius, .theta. is
the angle between the electric field and the normal to the cell
surface, and f is a geometric factor that is typically around 3/2.
This implies that to form pores at the poles of the cell the
electric field should be about 1 kV/cm for a cell with radius of 8
microns.
[0070] With this electroporation device, the application of a 5 V
potential difference between the top and bottom plates results in
an electric field within the cell flow layer of about 1 kV/cm given
the electric field strengths and flow layer depths described. The
preferable range of applied voltages is between 1 V and 100 V. If
the patterned electrodes are 2.5 cm by 0.5 cm in size, then for a 5
V applied potential, a current of about 0.17 A is generated and a
power of 0.87 J/s is dissipated. This amount of power would
increase the temperature of pure water in a device with dimensions
5 cm by 2.5 cm by 0.01 cm by 1.7 degrees C./s, assuming that no
heat is dissipated through the boundary. The source for the applied
voltage can be from a battery with a fixed voltage or a battery
used in conjunction with a resistive voltage divider to enable the
voltage to be varied over the selected range. Commercial voltage
supplies are also readily available to provide selected voltages in
the range of 1 V to 100 V. An alternative electrode size example
includes electrodes with dimensions of 2.5 cm by 0.05 cm in size,
then for a 5 V applied potential, a current of about 0.017 A is
generated and a power of 0.087 J/s is dissipated. This amount of
power would increase the temperature of pure water in a device with
dimensions 5 cm by 2.5 cm by 0.01 cm by 0.17 degrees C./s. In a
typical experiment, cells at a density of 1.0.times.10.sup.7/mL are
flowed through the chip at a volumetric rate of approximately 1.5
mL/min, with a preferable range between 0.01 and 100 mL/min. The
nominal flow rate of 1.5 mL/min results in an average linear flow
velocity of 1.0 cm/s. At this velocity, cells are subject to the
electric field from an electrode that is 2.5 cm by 0.5 cm in width
and length for 0.5 s. Assuming Hele-Shaw flow, the pressure
difference across the input and output of the chip is about 40 atm.
It is important to note that during the approximately 0.5 s that
cells are subject to the electric field, that the plasmid DNA is
electrophoretically driven toward the cell flow layer, assuming
that the plasmid is in the lower sheath flow and that the top
electrode is held at a higher voltage than the bottom electrode.
Assuming a DNA mobility of 4.times.10.sup.-4 cm.sup.2/Vs, the
average time that it takes a DNA molecule to move half-way through
a distance of 25 microns (the typical depth of the sheath flow
layer containing the plasmid) is 0.34 s. A DNA molecule that
reaches the cell flow layer is driven across it in about 10 ms.
[0071] Another important timescale is the cell sedimentation time
for falling a distance of one-half of the cell flow layer
thickness. Again assuming a cell radius of 8 microns, a difference
in density between a cell and the surrounding fluid of 0.07
g/cm.sup.3, and that the hydrodynamic friction coefficient of a
cell is 6.pi..eta.R, where .eta. is the buffer viscosity
(approximately 0.001 Pa, but may be higher with additive chemicals
such as sucrose), the time to drop a distance of 25 microns is
approximately 0.4 s. And the time for a typical salt ion, such as
Na or K, to diffuse a distance of 25 microns is 0.6 s. This
indicates that the flow layers remain laminar (and retain their
respective conductivities) for the time it takes the cells to cross
the electrode region when the patterned electrodes are about 2.5 cm
by 0.5 cm in width and length.
[0072] Following the electroporation of a given volume of cells the
electroporation efficiency and cell viability are determined by
phase contrast and static fluorescent imaging, and sometimes by
flow cytometry. After the cells are electroporated with the
GFP-expressing plasmid in the flow chip, the cells are collected
and transferred to a 96 or 24 well plate with appropriate cell
medium, such as DMEM. The cells are cultured at 37.degree. C. in an
incubator with 5% CO.sub.2 for 1, 6, 12, 24, or 48 hours. The cells
are centrifuged at 300 g for 5 min and the aspirant is discarded.
The cells are washed with PBS and this process is repeated.
Following this, the cells are stained with propidium iodide
(Invitrogen) at a concentration of approximately 1 microgram/mL.
The cells are incubated in the dark for 15 min and then optically
examined by phase contrast under fluorescent filters. A standard
GFP filter set is used to determine the fraction of cells that have
been electroporated with the plasmid. A filter set with excitation
at 488 nm and emission at approximately 620 nm is used to determine
the dead cells that have been permeated by propidium iodide.
Several images can be acquired at different locations to improve
the statistics of the electroporation efficiency and the cell
viability. The cells may also be examined by flow cytometry to
determine the fraction that has been electroporated as identified
by a green fluorescent signal and the fraction that are dead as
identified by uptake of propidium iodide and a red fluorescent
signal.
[0073] Thus the described chip can reliably be used to
electroporate a large number of mammalian or bacterial cells at
high efficiency and with low cell death in a short amount of time.
The cells can be transfected with plasmid DNA that can be
transcribed into a protein that is therapeutic for a disease. The
cells can be transfected with mRNA that is likewise transcribed
into a protein that is necessary for improving the health of the
cell or that can be harvested for other medical use, such as
production of antibodies. The cells can also be transfected with
purified Cas9 protein, or another DNA guided nuclease, and
synthetic guide RNA molecules, termed ribonucleoproteins, in order
to efficiently edit a genomic site that is deleterious.
[0074] The method outlined in Example 1 can be used to
electroporate a variety of different mammalian cell types
including: CHO, Hela, T-cells, CD8+, CD4+, CD3+, PBMC, Huh-7,
Renca, NIH 3T3, Primary Fibroblasts, hMSCs, K562, Vero, HEK 293,
A549, B16, BHK-21, C2C12, C6, CaCo-2, CAP-T, COS-1, Cos-7, CV-1,
DLD-1, H1299, Hep G2, HOS, Jurkat, L5278Y, LNCaP, MCF7, MDA-MB-231,
MDCK, Mesenchymal Stem Cells, Min-6, Neuro2a, NIH3T3L1, NSO,
Panc-1, PC12, PC-3, RBL, RLE, SF21, SF9, SH-SY5Y, SK-MES-1,
SK-N-SH, SL3, SW403, THP-1, U205, U937.
[0075] The method outlined in Example 1 can be used to
electroporate a variety of different types of molecules to any
mammalian cell including: DNA, RNA, mRNA, siRNA, miRNA, other
nucleic acids, proteins, peptides, enzymes, metabolites, membrane
impermeable drugs, cryoprotectants, exogenous organelles, molecular
probes, nanoparticles, lipids, carbohydrates, small molecules, and
complexes of proteins with nucleic acids (like CAS9-sgRNA). While
the method outlined in Example 1 relies on an electric field to
deliver charged nucleic acid molecules to electroporated cells, the
method also suffices to electroporate neutral molecules where
diffusive motion is sufficient for the delivery.
[0076] Although various embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the disclosure and these are therefore considered to be
within the scope of the disclosure as defined in the claims which
follow.
* * * * *